Parental early life environments drive transgenerational plasticity of offspring metabolism in a freshwater fish (Danio rerio)

Parental experiences can lead to changes in offspring phenotypes through transgenerational plasticity (TGP). TGP is expected to play a role in improving the responses of offspring to changes in climate, but little is known about how the early lives of parents influence offspring TGP. Here, we use a model organism, zebrafish (Danio rerio), to contrast the effects of early and later life parental thermal environments on offspring routine metabolism. To accomplish this, we exposed both parents to either constant optimal (27°C) or environmentally realistic diel fluctuating (22–32°C) temperatures during early (embryonic and larval) and later (juvenile and adult) life in a factorial design. We found significant reduction of routine metabolic rates (greater than 20%) at stressful temperatures (22°C and 32°C) after biparental early life exposure to fluctuating temperatures, but little effect of later life parental temperatures on offspring metabolism. This reduction reflects metabolic compensation and is expected to enhance offspring body sizes under stressful temperatures. These changes occur over and above the effects of parental environments on egg size, suggesting alternate non-genetic mechanisms influenced offspring metabolic rates.


Background
The experiences of parents can impact their offspring through a suite of mechanisms that transcend genetic inheritance, including parental care, offspring provisioning and epigenetic facilitation [1,2].These non-genetic parental effects, collectively termed 'transgenerational plasticity' (TGP), have drawn considerable attention for their role in shaping offspring phenotypic variation and fitness [3,4] and potential to facilitate rapid, beneficial responses to changing environments [2,5,6].
Temperature is a key driver of phenotypic plasticity in ectotherms [7], and continued increases in global thermal means and variance are expected to negatively impact many aspects of organismal performance, including body size via increased metabolic demands as temperatures rise [8][9][10].Yet, through TGP, parental exposure to warm temperatures can improve outcomes for offspring experiencing warm temperatures themselves, relative to offspring whose parents received no such cue ('anticipatory parental effects' [11]; reviewed in [2]).For example, lifelong or reproductive parental acclimation to warm, constant temperatures increases the relative growth rates and body sizes of warm-incubated offspring in fishes [12][13][14][15][16][17] and is associated with epigenetic increases in offspring metabolic efficiency [12,18].However, no studies to date have examined TGP in response to variable temperatures, which better reflect natural or predicted thermal conditions.Thermal variability and periodic heating events are expected to intensify with climate change [19,20], and constant versus variable temperatures often produce different phenotypic effects, even when sharing thermal means [21,22].There is therefore an urgent need to understand how TGP may manifest under ecologically realistic thermal variability [2,23].
The occurrence and strength of TGP can also be influenced by the ontogenetic timing during which parents experience thermal stressors, and most studies have investigated parental exposures during sexual maturity and reproduction [2,24].However, embryos and larvae may be especially environmentally sensitive, and cellular/molecular changes during early development can cascade into large effects on parental and subsequent offspring phenotypes [6,25,26].Moreover, beneficial TGP is expected to occur when cues experienced by parents supply accurate information about offspring environments; the windows of parental sensitivity should thus vary depending on the ecology of focal organisms [1,3,11].In some cases, such as when juvenile and adult habitat use differs, parental early life environments may be better predictors of offspring environments [27,28].
Few studies have compared the transgenerational effects of pre-and post-maturity parental thermal experiences ( [2,24]; but see: [29]).In fishes, pre-maturity parental exposure to warm, constant temperatures appears to have beneficial effects on offspring in warm conditions, enhancing body size [30] and aerobic scope [31,32].Although these studies support the existence of early ontogentic critical windows for TGP, they do not delineate the influence of the earliest parental life stages from later juvenile development.The sensitivity of parental development between fertilization and the juvenile stage is thus of particular interest, especially as these stages are highly responsive to environmentally induced epigenetic modifications [2,25].
Here, we use zebrafish (Danio rerio) as a model to investigate how the timing of biparental thermal exposure influences offspring metabolism, by comparing the effects of constant versus challenging diel variable thermal treatments in a 1-year long factorial experiment.We delineate parental 'early life' as pre-metamorphic embryonic and larval stages (day 0 to 29), in contrast to 'later' post-metamorphic juvenile and adult stages (day 30+).We chose this delineation because premetamorphosis is a candidate window for epigenetic effects [25], defined by significant tissue differentiation in teleost fishes [33].Moreover, the earliest life stages of wild zebrafish occur during the distinctly thermally variable monsoon season (approx.12-31°C) [34,35].Zebrafish parental early life environments may therefore act as better cues for young offspring than later life environments (i.e.given environmental autocorrelation between monsoon seasons) [1,28].We reared parents in thermal treatments for 1 year, simulating their annual life cycle [34], and tested for TGP in routine metabolic rate (RMR) of 1-day old offspring at stressful temperatures (22°C and 32°C).We measured this trait because plasticityinduced reductions in RMR can improve subsequent juvenile growth in stressful thermal conditions, [12,18,36], and juvenile size is a fitness-related trait in teleost fishes [37].

Methods (a) Fish husbandry and thermal treatments
We collected parental generation (F 1 ) zebrafish embryos from three non-sibling matings (families) of wild-type-AB fish within 3 h post-fertilization (hpf ) from the Dalhousie Zebrafish Core.Grandparental source fish (F 0 ) were kept in control standard zebrafish laboratory conditions at 27°C (electronic supplementary material).
F 1 embryos were brought to the Dalhousie University Aquatron and each family was divided into two thermal treatment groups (figure 1): a Constant 27°C, which reflects a thermal optimum for reproduction and growth in zebrafish [34,35], and a Fluctuating temperature that varied sinusoidally on a diel basis (22-32°C; figure 1).This regime was intended to reflect natural thermal variability [34], while exposing fish to transient stressful temperatures, minimizing constant temperature-induced pathologies [21,34,[38][39][40].
F 1 embryos and larvae were reared in their initial 'Early Parental Temperature' treatment (Constant or Fluctuating) for 29 dpf (days post-fertilization).At the onset of the juvenile stage (30 dpf), fish were equally split into Constant or Fluctuating temperatures in groups of 12-15, and held in these conditions through sexual maturity (90 dpf) until 1 year of age, establishing a 'Later Parental Temperature' treatment (figure 1).In total, there were four groups representing factorial combinations of Early and Later Parental Temperature treatments, with six replicate tanks per group, each with two replicates per family.At 1 year of age, sibling F 1 fish were bred within each treatment to produce F 2 embryos, which were kept at 27°C for 24 h (figure 1; electronic supplementary material).

(b) Embryo metabolism
We measured RMRs of 24 hpf F 2 embryos at two acute Test Temperatures (22 and 32°C) representing the thermal extremes at which zebrafish develop normally [35].We used a Microplate Respirometry System (Loligo Systems) with two 24-well, 80 µl microplates run in parallel.Microplates were calibrated to manufacturer specifications the previous night.We photographed individual embryos under a dissecting scope and placed embryos into wells randomly assigned by MicroResp software (Loligo Systems), with four blanks (electronic supplementary material).Microplates were then set to the two Test Temperatures, and the entire system was placed on an orbital shaker on the lowest setting, reducing oxygen stratification.We recorded oxygen consumption in each microplate for at least 3 h.We repeated measurements across six trials, with each trial day consisting of offspring from one of the three F 1 families, and each of the four parental thermal treatment combinations.

(c) Statistical analyses
We used MicroResp software to estimate embryo RMR in normoxia (80-100% oxygen saturation).We selected an r 2 value of ≥0.8 for linear estimations of oxygen consumption (MO 2 ) rates, and applied automatic correction for background respiration from blanks.
We measured F 1 embryo egg diameter from photographs as a proxy for offspring size.We could not consistently measure yolk diameters, as yolks were occasionally obscured by embryos; however, we previously showed a strong positive correlation between yolk and egg diameters across thermal treatments [41].Upon reviewing images, we excluded embryos that exhibited deformity (e.g.kyphosis) or had visible tears in their chorions.
We used Bayesian generalized linear mixed models to analyse data using the brms package (v.2.19.0 [42]) in R (v. 4.2.2).These models estimated the causal effect of predictors on offspring RMR.For Model 1, we estimated the effects of 'Early' (0-29 dpf ) and 'Later' (30 + dpf ) Parental Temperatures (Constant or Fluctuating) as well as the acute 'Test' Temperature (22 or 32°C) on log-transformed embryo RMR [43].We included interactions between 'Test' Temperature and each Parental Temperature treatment, to test for differences in TGP thermal sensitivity.We included 'Trial' as a random intercept to account for within-trial interdependence (e.g.due to the sum of differences in daily calibration and family level effects).To test whether changes in offspring RMR were mediated via changes to egg size, we also fitted Model 2, correcting for 'Egg Size' (diameter).Using two separate models allowed us to avoid overcontrol bias [40] in our estimation of 'Later' Parental Temperature in Model 1 as this treatment impacts egg size [41].We interpreted non-negligible effects of Early or Later Parental Temperatures on offspring RMR as evidence of TGP, and interactions between Parental Temperatures and Test Temperatures as differences in the thermal sensitivity of TGP between Test Temperatures.Model specifications are detailed in electronic supplementary material.

Results
Median results for metabolism and egg size, with individual data circled, are illustrated in figure 2a,b, and posterior effect sizes for Model 1 are illustrated in figure 2c.Briefly, mean effect sizes further from 0 indicate stronger effects of predictors on offspring RMR, and the greater the overlap of uncertainty intervals (UIs) with 0, the less certainty in estimates.
There was a strong influence of egg diameter (figure 2b) on RMR, such that a 1 mm increase in diameter was predicted to lead to a 70% increase in RMR, with wide variation in the posterior distribution (Model 2; 90% UIs:

Discussion
Although TGP is an important mechanism through which organisms can modify offspring phenotypes to withstand climatic stressors, we have few empirical data describing the role of parents' early life thermal experiences on offspring responses [24].Here, we show that biparental thermal experiences of F 1 zebrafish during their first month of life strongly affected F 2 offspring embryonic routine metabolic rates when exposed to stressful temperatures.Indeed, metabolic rates were over 20% lower at thermal extremes when parents were initially reared in challenging, thermally variable conditions compared to optimal, constant early life conditions.
Anthropogenic climate warming has increased ectotherm metabolic rates worldwide [10,36], leading to increases in energetic demands and ultimately imposing constraints on adult body sizes [8,9].Although interpretations of plasticity-induced reductions in metabolism are context dependent [44], lower metabolic rates are predicted to confer growth benefits under stressful thermal environments [18,36].Although later offspring body sizes were not measured here, other studies rearing fish in warm temperatures have explicitly linked lower routine metabolic rates to improved growth rates via TGP [12,18] and within-generation plasticity [45].Therefore, we interpret the reduction in embryonic metabolic rate as beneficial metabolic compensation, which could ameliorate negative impacts of thermal stressors on body size as offspring develop [12,18,44,45].
We also tested whether these reductions in metabolic rate were mediated via direct reductions in egg size, given that metabolic rates positively scale with organismal size [46] and later life fluctuating temperatures reduce both parental body sizes and egg provisioning in zebrafish [41].By correcting for differences in egg size, the small decrease in offspring RMR owing to fluctuating later life parental temperatures disappeared.This may reflect a condition-transfer effect [47], in which constraints on parental energy expenditure during later life led to a reduction in egg size, and thus offspring RMR [11,41].By contrast, the large reduction in RMR caused by fluctuating early parental temperatures occurred over and above effects on egg size.Instead, it is likely that this metabolic compensation was facilitated through alternative epigenetic mechanisms established during parental early life [25].For example, beneficial TGP to warm temperatures has been shown to be mediated through differential gene expression, including the upregulation of genes associated with mitochondrial activity and energy production [15,32,48].Other potential mechanisms include methylation and histone modifications in germline cells during early parental development [6,24,25].
In this experiment, we also found a modest interaction between early parental temperatures and acute test temperatures.Contrary to previous findings, however, we found slightly less metabolic reduction at the hotter versus the cooler offspring test temperature for offspring from early fluctuating temperature parents [12,30].It is possible that the scope for thermal sensitivity of TGP was reduced in the acute hot test temperature because of physiological limitations, as 32°C is the upper thermal limit for the normal development of zebrafish embryos [35].
Overall, we found that there is strong TGP in offspring routine metabolic rate in response to ecologically realistic thermal variability experienced during parental early life, but a negligible influence of later life adult thermal experiences.These data highlight the importance of studying how the timing of stressors influences plasticity, and show that early life parental environments can generate significant phenotypic variation in subsequent generations.A goal of future studies will be to understand the long-term consequences of these changes.

Figure 2 .
Figure 2. Transgenerational plasticity in offspring metabolism of F 1 zebrafish (Danio rerio) exposed to Constant ('C', 27°C) or diel Fluctuating ('F', 22-32°C) temperatures during Early Parental development (0-29 days, first letter), Later Parental development (30 + days, second letter), or both.(a) F 2 offspring routine metabolic rates (RMR) tested at Cool (teal; 22°C) or Hot (red; 32°C) thermal extremes.(b) Offspring egg diameter.(c) Posterior distributions of effect sizes of Parental Temperatures, Test Temperature and their interactions on F 2 offspring RMR.Posterior means are represented by thin dark lines at the mean of each distribution; 90% and 50% uncertainty intervals are represented by increasingly light shading.